1 Scripts

The code used for analysis is located on GitHub and compiled together here.

2 Quality control

2.1 Calling nuclei barcodes

  • First, we used Cell Ranger ARC called cell barcodes – algorithm described here. 14,301 barcodes out of 2,215,183.
  • Second, because the Cell Ranger ARC cell calling algorithm is very permissive to barcodes with very low counts (i.e., a minimum of a single count in each library), barcodes were additionally filtered to a low count threshold in both the ATAC and RNA libraries based on the clearly defined population of cells in the RNA and ATAC count scatterplot. Additionally, barcodes with more than 5% of RNA reads mapping to genes on the mitochondrial genome were excluded. A total of 886 barcodes were filtered out for 13,145 left.
  • Last, multiplets were filtered out using two independent methods, relying on either the ATAC or RNA libraries to call multiplets. AMULET relies on the assumption that in snATAC-seq of diploids there should be at most two overlapping fragments with the same cell barcode. The presence of more than two overlapping fragments is a potential indication of a multiplet. Doublets were also identified using the RNA-seq libraries with DoubletFinder. After removing the 1,103 multiplet barcodes, there were still two barcodes that had substantially higher number of RNA reads after all filtering (i.e., n_Count_RNA around 12-13k when the next highest are around 6-7k). So I removed those two for a final number of 12,040 high quality nuceli barcodes.

2.2 Knee plot

2.3 Scatter plot of counts

Cells after all filtering

Cell Ranger ARC only

And low count threshold

2.4 Violin QC plots

After filtering

Before filtering

2.5 Multiplets

Mapped onto UMAP projection

Counts

Count histograms

Multiplets

After filtering

2.6 QC metrics per sample

Number of cells

ATAC counts

RNA counts

TSS Enrichment

FRiP

FRiT

2.7 QC metrics on UMAP

ATAC counts

RNA counts

TSS enrichment

Mito percent

FRiP

FRiT

3 Seurat

3.1 Clusters

Cells per cluster

Acclimation clusters

Joint UMAP

Split

Together

RNA-only UMAP

Split

Together

ATAC-only UMAP

Split

Together

3.2 Annotation

3.2.1 Annotation results

cluster annot prop_overlap pval padj
0 ventral epidermis primordium 0.0532787 0.0000000 0.0000000
0 dorsal epidermis primordium 0.0532787 0.0000000 0.0000000
0 dorsal ectoderm primordium 0.0368852 0.0000002 0.0000159
0 embryonic dorsal epidermis 0.0491803 0.0000007 0.0000484
0 tracheal primordium 0.0245902 0.0000098 0.0004196
0 embryonic ventral epidermis 0.0409836 0.0000121 0.0005023
0 dorsal epidermis anlage 0.0122951 0.0000838 0.0026510
0 ventral ectoderm anlage in statu nascendi 0.0204918 0.0003854 0.0098415
0 subset 0.0245902 0.0005109 0.0121885
0 foregut primordium 0.0245902 0.0005463 0.0126547
0 clypeo-labral primordium 0.0163934 0.0006188 0.0141685
0 hindgut proper primordium 0.0286885 0.0008120 0.0179733
0 ventral ectoderm primordium 0.0204918 0.0010421 0.0218520
0 embryonic/larval tracheal system 0.0204918 0.0013134 0.0268347
0 embryonic epipharynx 0.0204918 0.0018848 0.0362357
0 ventral epidermis primordium P2 0.0081967 0.0020118 0.0379853
0 atrium 0.0163934 0.0026519 0.0465421
1 trunk mesoderm primordium 0.0585106 0.0000053 0.0002496
1 muscle system primordium 0.0265957 0.0001090 0.0033143
1 somatic muscle primordium 0.0265957 0.0004709 0.0115811
1 longitudinal visceral mesoderm primordium 0.0159574 0.0013004 0.0267054
2 ventral nerve cord primordium 0.0388350 0.0000317 0.0011599
2 brain primordium 0.0388350 0.0000503 0.0016699
2 ventral nerve cord primordium P3 0.0258900 0.0002285 0.0063667
2 ventral ectoderm primordium P2 0.0226537 0.0002856 0.0076374
2 no staining 0.0194175 0.0014403 0.0289806
2 embryonic brain 0.0388350 0.0020681 0.0388641
2 central brain primordium P3 0.0129450 0.0022350 0.0412232
3 posterior midgut primordium 0.0996785 0.0000000 0.0000000
3 anterior midgut primordium 0.0932476 0.0000000 0.0000000
3 posterior endoderm primordium 0.0707395 0.0000000 0.0000000
3 anterior endoderm primordium 0.0675241 0.0000000 0.0000000
3 embryonic midgut 0.0868167 0.0000000 0.0000000
3 embryonic/larval muscle system 0.0546624 0.0000000 0.0000000
3 trunk mesoderm primordium 0.0610932 0.0000000 0.0000002
3 posterior endoderm primordium P2 0.0450161 0.0000000 0.0000005
3 somatic muscle primordium 0.0353698 0.0000000 0.0000013
3 embryonic/larval visceral muscle 0.0385852 0.0000000 0.0000033
3 dorsal prothoracic pharyngeal muscle 0.0385852 0.0000002 0.0000130
3 dorsal pharyngeal muscle primordium 0.0257235 0.0000004 0.0000302
3 visceral muscle primordium 0.0289389 0.0000010 0.0000640
3 anterior endoderm anlage 0.0353698 0.0000011 0.0000647
3 embryonic/larval somatic muscle 0.0321543 0.0000011 0.0000650
3 muscle system primordium 0.0257235 0.0000012 0.0000718
3 embryonic anal pad 0.0289389 0.0000033 0.0001646
3 salivary gland primordium 0.0192926 0.0000039 0.0001888
3 head mesoderm primordium P2 0.0353698 0.0000113 0.0004780
3 head mesoderm primordium 0.0257235 0.0000225 0.0008548
3 cellular blastoderm 0.0385852 0.0000415 0.0014407
3 embryonic hindgut 0.0385852 0.0000448 0.0015240
3 maternal 0.1028939 0.0000890 0.0027922
3 salivary gland body primordium 0.0192926 0.0002808 0.0076106
3 longitudinal visceral mesoderm primordium 0.0128617 0.0004456 0.0110955
3 embryonic/larval fat body 0.0192926 0.0005423 0.0126349
3 foregut primordium 0.0192926 0.0018918 0.0362357
3 trunk mesoderm anlage 0.0064309 0.0019156 0.0365160
3 head mesoderm primordium P4 0.0225080 0.0021003 0.0392842
3 ubiquitous 0.0643087 0.0021180 0.0394311
4 ventral epidermis primordium 0.0437158 0.0000002 0.0000168
4 dorsal ectoderm primordium 0.0382514 0.0000036 0.0001738
4 dorsal epidermis primordium 0.0382514 0.0000095 0.0004122
4 tracheal primordium 0.0273224 0.0000335 0.0012120
4 embryonic ventral epidermis 0.0437158 0.0000569 0.0018588
4 embryonic dorsal epidermis 0.0437158 0.0001155 0.0034612
4 embryonic/larval tracheal system 0.0273224 0.0003611 0.0093414
4 subset 0.0273224 0.0009376 0.0204129
4 hindgut proper primordium 0.0327869 0.0009616 0.0205963
4 ventral epidermis primordium P2 0.0109290 0.0011416 0.0236881
4 embryonic foregut 0.0273224 0.0016159 0.0318710
5 muscle system primordium 0.0307692 0.0003132 0.0082638
5 visceral muscle primordium 0.0307692 0.0009039 0.0197870
5 longitudinal visceral muscle fibers 0.0153846 0.0015871 0.0314581
6 tracheal primordium 0.0508475 0.0000000 0.0000000
6 embryonic/larval tracheal system 0.0451977 0.0000001 0.0000129
6 dorsal epidermis primordium 0.0451977 0.0000006 0.0000439
6 dorsal ectoderm primordium 0.0395480 0.0000029 0.0001485
6 embryonic ventral epidermis 0.0508475 0.0000059 0.0002705
6 embryonic dorsal epidermis 0.0508475 0.0000133 0.0005317
6 ventral epidermis primordium 0.0338983 0.0000303 0.0011268
6 primary segmental branch specific anlage 0.0112994 0.0001209 0.0035668
6 embryonic salivary gland duct 0.0169492 0.0001680 0.0048494
6 visceral branch specific anlage 0.0112994 0.0001842 0.0052796
6 dorsal trunk specific anlage 0.0112994 0.0005672 0.0130612
6 dorsal epidermis anlage 0.0112994 0.0017218 0.0334610
6 embryonic salivary gland common duct 0.0112994 0.0019356 0.0367202
6 embryonic head epidermis 0.0282486 0.0023524 0.0425996
7 ventral nerve cord primordium P3 0.0448980 0.0000001 0.0000074
7 brain primordium 0.0571429 0.0000001 0.0000117
7 ventral nerve cord primordium 0.0530612 0.0000005 0.0000369
7 embryonic brain 0.0653061 0.0000008 0.0000506
7 procephalic ectoderm primordium 0.0367347 0.0000020 0.0001084
7 procephalic ectoderm anlage 0.0326531 0.0000030 0.0001555
7 dorsal ectoderm anlage in statu nascendi 0.0285714 0.0000067 0.0003047
7 ventral nerve cord anlage 0.0244898 0.0000131 0.0005264
7 ventral ectoderm primordium 0.0285714 0.0000131 0.0005264
7 procephalic ectoderm anlage in statu nascendi 0.0244898 0.0000395 0.0013943
7 ventral nerve cord 0.0489796 0.0002790 0.0076106
7 ventral ectoderm anlage in statu nascendi 0.0204082 0.0003926 0.0099626
7 head mesoderm anlage in statu nascendi 0.0122449 0.0004129 0.0104104
7 amnioserosa anlage in statu nascendi 0.0122449 0.0004578 0.0113295
7 ectoderm anlage in statu nascendi 0.0122449 0.0004815 0.0117678
7 ubiquitous 0.0734694 0.0004957 0.0119689
7 subset 0.0244898 0.0005220 0.0123785
7 cellular blastoderm 0.0367347 0.0005275 0.0123939
7 central brain primordium P3 0.0163265 0.0009581 0.0205963
7 head epidermis dorsal anlage in statu nascendi 0.0081633 0.0011976 0.0247220
7 anlage in statu nascendi 0.0163265 0.0016566 0.0325115
7 anterior endoderm anlage in statu nascendi 0.0122449 0.0024786 0.0441834
7 yolk nuclei 0.0204082 0.0024842 0.0441834
7 maternal 0.0938776 0.0025898 0.0458062
8 plasmatocytes anlage 0.0365854 0.0000000 0.0000000
8 head mesoderm primordium P2 0.0560976 0.0000000 0.0000000
8 yolk nuclei 0.0439024 0.0000000 0.0000000
8 amnioserosa 0.0414634 0.0000000 0.0000000
8 plasmatocytes 0.0219512 0.0000000 0.0000001
8 garland cell primordium 0.0219512 0.0000000 0.0000001
8 head mesoderm primordium P4 0.0365854 0.0000000 0.0000019
8 embryonic salivary gland 0.0243902 0.0000004 0.0000286
8 posterior endoderm primordium P2 0.0317073 0.0000009 0.0000579
8 embryonic/larval garland cell 0.0195122 0.0000012 0.0000720
8 maternal 0.1073171 0.0000013 0.0000752
8 salivary gland body primordium 0.0219512 0.0000031 0.0001562
8 ubiquitous 0.0756098 0.0000034 0.0001658
8 crystal cell 0.0146341 0.0000082 0.0003637
8 embryonic/larval fat body 0.0219512 0.0000085 0.0003702
8 anterior endoderm anlage 0.0268293 0.0000145 0.0005656
8 posterior endoderm primordium 0.0317073 0.0000236 0.0008887
8 procrystal cell 0.0121951 0.0000430 0.0014782
8 anterior endoderm primordium 0.0292683 0.0000697 0.0022403
8 embryonic midgut 0.0487805 0.0000829 0.0026429
8 head mesoderm anlage in statu nascendi 0.0097561 0.0001014 0.0031303
8 cellular blastoderm 0.0317073 0.0001462 0.0042511
8 posterior midgut primordium 0.0390244 0.0002203 0.0062258
8 salivary gland primordium 0.0121951 0.0002226 0.0062441
8 trunk mesoderm primordium 0.0317073 0.0004389 0.0109985
8 embryonic dorsal epidermis 0.0268293 0.0004901 0.0119068
8 apically cleared 0.0097561 0.0008015 0.0178385
8 mesoderm anlage in statu nascendi 0.0097561 0.0010420 0.0218520
8 anterior endoderm anlage in statu nascendi 0.0097561 0.0010801 0.0225286
8 dorsal epidermis primordium 0.0170732 0.0013450 0.0272322
8 anterior midgut primordium 0.0341463 0.0013466 0.0272322
8 trunk mesoderm primordium P2 0.0219512 0.0015033 0.0300668
8 posterior endoderm anlage in statu nascendi 0.0097561 0.0017201 0.0334610
8 embryonic/larval tracheal system 0.0146341 0.0024406 0.0439969
8 ventral epidermis primordium 0.0146341 0.0025984 0.0458062
9 amnioserosa 0.0416667 0.0000000 0.0000000
9 yolk nuclei 0.0321970 0.0000000 0.0000000
9 embryonic salivary gland 0.0284091 0.0000000 0.0000000
9 embryonic dorsal epidermis 0.0416667 0.0000000 0.0000001
9 embryonic ventral epidermis 0.0378788 0.0000000 0.0000003
9 salivary gland body primordium 0.0246212 0.0000000 0.0000006
9 maternal 0.1098485 0.0000000 0.0000012
9 dorsal ectoderm primordium 0.0227273 0.0000004 0.0000275
9 embryonic epipharynx 0.0227273 0.0000005 0.0000398
9 embryonic midgut 0.0530303 0.0000007 0.0000484
9 embryonic hypopharynx 0.0227273 0.0000009 0.0000597
9 embryonic proventriculus 0.0227273 0.0000013 0.0000746
9 embryonic/larval tracheal system 0.0208333 0.0000017 0.0000917
9 dorsal epidermis primordium 0.0227273 0.0000017 0.0000917
9 subset 0.0227273 0.0000021 0.0001106
9 head mesoderm primordium P2 0.0284091 0.0000045 0.0002118
9 posterior midgut primordium 0.0416667 0.0000059 0.0002705
9 salivary gland primordium 0.0132576 0.0000071 0.0003163
9 ventral epidermis primordium 0.0189394 0.0000128 0.0005244
9 posterior endoderm primordium P2 0.0246212 0.0000136 0.0005350
9 dorsal ectoderm anlage in statu nascendi 0.0170455 0.0000212 0.0008104
9 garland cell primordium 0.0113636 0.0000305 0.0011268
9 anterior midgut primordium 0.0378788 0.0000353 0.0012685
9 ventral ectoderm primordium 0.0170455 0.0000470 0.0015869
9 embryonic hindgut 0.0303030 0.0000495 0.0016569
9 anterior endoderm primordium 0.0265152 0.0000520 0.0017106
9 apoptotic amnioserosa 0.0075758 0.0000578 0.0018711
9 ubiquitous 0.0625000 0.0000939 0.0029234
9 embryonic proventriculus outer layer 0.0075758 0.0001035 0.0031718
9 embryonic head epidermis 0.0208333 0.0001128 0.0034036
9 hindgut proper primordium 0.0227273 0.0001184 0.0035205
9 anterior endoderm anlage 0.0208333 0.0001389 0.0040680
9 embryonic foregut 0.0189394 0.0001873 0.0053299
9 atrium 0.0132576 0.0002623 0.0072568
9 head mesoderm anlage in statu nascendi 0.0075758 0.0002658 0.0073031
9 posterior endoderm primordium 0.0246212 0.0002910 0.0077299
9 ventral ectoderm primordium P2 0.0170455 0.0003336 0.0086855
9 foregut primordium 0.0170455 0.0003729 0.0095849
9 embryonic/larval garland cell 0.0113636 0.0005089 0.0121885
9 trunk mesoderm anlage in statu nascendi 0.0075758 0.0005289 0.0123939
9 procephalic ectoderm anlage 0.0151515 0.0006406 0.0145830
9 tracheal primordium 0.0113636 0.0006581 0.0148964
9 procephalon primordium 0.0056818 0.0007305 0.0164421
9 embryonic salivary gland body 0.0094697 0.0007527 0.0168459
9 head mesoderm primordium P4 0.0189394 0.0009603 0.0205963
9 plasmatocytes anlage 0.0094697 0.0009879 0.0210473
9 no staining 0.0946970 0.0010414 0.0218520
9 trunk mesoderm primordium 0.0265152 0.0015094 0.0300668
9 gnathal primordium 0.0056818 0.0017378 0.0336094
9 ventral ectoderm anlage in statu nascendi 0.0113636 0.0021914 0.0406077
9 clypeolabrum primordium P2 0.0056818 0.0022465 0.0412449
9 trunk mesoderm primordium P2 0.0189394 0.0024784 0.0441834
10 maternal 0.2123711 0.0000000 0.0000000
10 ubiquitous 0.1567010 0.0000000 0.0000000
10 trunk mesoderm primordium 0.0597938 0.0000000 0.0000000
10 embryonic midgut 0.0762887 0.0000000 0.0000000
10 faint ubiquitous 0.0701031 0.0000000 0.0000000
10 embryonic/larval muscle system 0.0474227 0.0000000 0.0000000
10 anterior midgut primordium 0.0577320 0.0000000 0.0000000
10 posterior midgut primordium 0.0577320 0.0000000 0.0000001
10 cellular blastoderm 0.0474227 0.0000000 0.0000001
10 head mesoderm primordium 0.0309278 0.0000000 0.0000001
10 trunk mesoderm primordium P2 0.0371134 0.0000000 0.0000005
10 anterior endoderm primordium 0.0391753 0.0000000 0.0000008
10 posterior endoderm primordium 0.0371134 0.0000001 0.0000067
10 anterior endoderm anlage 0.0309278 0.0000001 0.0000067
10 dorsal prothoracic pharyngeal muscle 0.0309278 0.0000001 0.0000074
10 somatic muscle primordium 0.0247423 0.0000001 0.0000117
10 posterior endoderm primordium P2 0.0309278 0.0000002 0.0000148
10 embryonic/larval visceral muscle 0.0268041 0.0000006 0.0000409
10 head mesoderm primordium P4 0.0288660 0.0000009 0.0000597
10 embryonic/larval somatic muscle 0.0247423 0.0000014 0.0000811
10 head mesoderm primordium P2 0.0309278 0.0000016 0.0000885
10 salivary gland body primordium 0.0185567 0.0000119 0.0004975
10 strong ubiquitous 0.0164948 0.0000209 0.0008080
10 embryonic brain 0.0412371 0.0000369 0.0013135
10 fat body specific anlage 0.0082474 0.0000416 0.0014407
10 brain primordium 0.0288660 0.0002837 0.0076357
10 ventral nerve cord 0.0371134 0.0003220 0.0084401
10 hindgut proper primordium 0.0206186 0.0009010 0.0197870
10 longitudinal visceral mesoderm primordium 0.0082474 0.0022860 0.0417766
10 embryonic hindgut 0.0247423 0.0023353 0.0424833

3.2.2 Manual annotations

cluster annot_manual top_3
0 ectoderm primordium ventral epidermis primordium; dorsal epidermis primordium; dorsal ectoderm primordium
1 mesoderm primordium trunk mesoderm primordium; muscle system primordium; somatic muscle primordium
2 ventral nerve cord primordium ventral nerve cord primordium; brain primordium; ventral nerve cord primordium P3
3 endoderm primordium posterior midgut primordium; anterior midgut primordium; posterior endoderm primordium
4 ectoderm primordium ventral epidermis primordium; dorsal ectoderm primordium; dorsal epidermis primordium
5 muscle system primordium muscle system primordium; visceral muscle primordium; longitudinal visceral muscle fibers
6 tracheal primordium tracheal primordium; embryonic/larval tracheal system; dorsal epidermis primordium
7 ventral nerve cord primordium ventral nerve cord primordium P3; brain primordium; ventral nerve cord primordium
8 plasmatocytes anlage plasmatocytes anlage; head mesoderm primordium P2; yolk nuclei
9 amnioserosa amnioserosa; yolk nuclei; embryonic salivary gland
10 unknown maternal; ubiquitous; trunk mesoderm primordium

Note: Cluster 11 is likely also unknown or ubiquitous, as it does not have an annotation that passes FDR..

Note that there are five types of callouts, including: note, warning, important, tip, and caution.